CA3106325A1 - Carbon dioxide bioconversion process - Google Patents
Carbon dioxide bioconversion process Download PDFInfo
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- CA3106325A1 CA3106325A1 CA3106325A CA3106325A CA3106325A1 CA 3106325 A1 CA3106325 A1 CA 3106325A1 CA 3106325 A CA3106325 A CA 3106325A CA 3106325 A CA3106325 A CA 3106325A CA 3106325 A1 CA3106325 A1 CA 3106325A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/52—Propionic acid; Butyric acids
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
A CO2 bioconversion process includes providing a CO2 containing substrate to a bioreactor, the CO2 containing substrate including about 5 to about 90 mole % CO2; and fermenting the CO2 containing substrate with an acetogenic bacteria carrying a sodium translocating ATPase. The medium including less than about 0.01 grams per liter yeast extract, less than about 0.01 grams per liter carbohydrate, a sodium ion concentration provided by a sodium ion feed rate of about 290 to about 8750 gg/gram of cells/minute, and a pH of about 4 to about 6.9.
Description
CARBON DIOXIDE BIOCONVERSION PROCESS
[0001] This application claims the benefit of U.S. Provisional. Application Nos.
62/716,083, filed August 8, 2018, 62/716,071, filed August 8, 2018, 62/716,053, filed August 8, 2018, 62/741,871, filed October 5, 2018, and 62/741,797, .filed October 5, 2018, all of which are incorporated in their entirety herein by reference.
[0001] This application claims the benefit of U.S. Provisional. Application Nos.
62/716,083, filed August 8, 2018, 62/716,071, filed August 8, 2018, 62/716,053, filed August 8, 2018, 62/741,871, filed October 5, 2018, and 62/741,797, .filed October 5, 2018, all of which are incorporated in their entirety herein by reference.
[0002] A process is provided for bioconversion of carbon dioxide. More specifically, the process includes providing a carbon dioxide containing gaseous stream to acetogenic bacteria. The process provides for high levels of carbon dioxide conversions and utilization of hydrogen.
BACKGROUND
BACKGROUND
[0003] Carbon dioxide generation occurs from natural processes as well as industrial processes that include .combustion of fossil fuels such as coal, oil and natural gas. Due in part to industrial processes, atmospheric carbon dioxide concentration continues to increase. These increases in carbon dioxide concentration may contribute to atmospheric changes which result in climate change and global warming. Carbon dioxide is difficult to utilize in biological processes because of its highly oxidized state.
[0004] In addition to carbon dioxide, many industrial processes also result in production of hydrogen. Hydrogen has a. high level of reducing potential. However, hydrogen is difficult to store and utilize due to its very flammable nature.
[0005] In view of large amount of carbon dioxide generated, there is a need for a bacterial fermentation system that can reduce a carbon dioxide footprint.
Further, there is a need for a fermentation system that can effectively utilize the reducing potential of hydrogen.
SUMMARY
Further, there is a need for a fermentation system that can effectively utilize the reducing potential of hydrogen.
SUMMARY
[0006] A process includes providing a gaseous substrate to a bioreactor. The gaseous substrate includes C07andcontains about 5 to about 90 mole % Ca). The process includes providing acetogenic bacteria to the bioreactor; providing sodium ions to the bioreactor through one or more sodium ion sources; and fermenting the gaseous substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids.
The acetogenic bacteria include a sodium translocating ATPase that is active during fermentation in the bioreactor. The fermentation broth includes less than about. 041 grams per liter yeast extract, less than about 0.01 grams per liter carbohydrate and a sodium ion concentration provided by a sodium feed rate of about 290 to about 8750 .1,1g/g of cells/minute. The fermentation broth is maintained at a pH in a range of about 4 to about .6.9.
The acetogenic bacteria include a sodium translocating ATPase that is active during fermentation in the bioreactor. The fermentation broth includes less than about. 041 grams per liter yeast extract, less than about 0.01 grams per liter carbohydrate and a sodium ion concentration provided by a sodium feed rate of about 290 to about 8750 .1,1g/g of cells/minute. The fermentation broth is maintained at a pH in a range of about 4 to about .6.9.
[0007] In another aspect, a process providing a gaseous substrate to a bioreactor. The gaseous substrate includes CO2 and 1-12 and contains about 5 to about 90 mole %
The process includes providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources; and fermenting the gaseous .substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids. The acetogenic bacteria includes a. sodium translocating ATPase that is.
active during fermentation in the bioreactor. The fermentation broth includes less than about 0.01 grams per liter yeast extract., less than about 0.01 grams per liter carbohydrate and a sodium ion concentration provided by a sodium feed rate of about 290 to about 8750 igig of.cells/minute.. The fermentation broth is maintained at a pH in a range of about4 to about 6.9.
The process includes providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources; and fermenting the gaseous .substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids. The acetogenic bacteria includes a. sodium translocating ATPase that is.
active during fermentation in the bioreactor. The fermentation broth includes less than about 0.01 grams per liter yeast extract., less than about 0.01 grams per liter carbohydrate and a sodium ion concentration provided by a sodium feed rate of about 290 to about 8750 igig of.cells/minute.. The fermentation broth is maintained at a pH in a range of about4 to about 6.9.
[0008] A composition includes one .or more of a source of NH4, P, LK, Fe, Niõ
Co, Se, Zn, W, or Mg; about 875 to. about 35,000 mg/L of a sodium ion source; about 0.009 to about 0.397 mg/L of a TV.10 source_ The composition includes less than about 0,01 grams.
per 'liter yeast extract, and less than about 0.01 grams per liter carbohydrates. The composition has a pH of about 4 to about 6.9..
BRIEF. DESCRIPTION OF FIGURES
Co, Se, Zn, W, or Mg; about 875 to. about 35,000 mg/L of a sodium ion source; about 0.009 to about 0.397 mg/L of a TV.10 source_ The composition includes less than about 0,01 grams.
per 'liter yeast extract, and less than about 0.01 grams per liter carbohydrates. The composition has a pH of about 4 to about 6.9..
BRIEF. DESCRIPTION OF FIGURES
[0009] So that the manner in which the above recited features of the present .disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may .be had .by reference to embodiments, some of which are 'illustrated in the appended drawings. It is to be noted, however, that the appended.
drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its .scope, for the disclosure may admit to other equally effective embodiments.
drawings illustrate only typical embodiments of this disclosure and are therefore not to be considered limiting of its .scope, for the disclosure may admit to other equally effective embodiments.
[0010] Figure I shows a. graph of CO2 conversion and H2 conversion by Acetobacteilum -wood in a bioreactor.
100111 Figure 2 illustrates acetic acid production by Acetobacterium wood/i.
[0012] Figure 3 describes growth of Acetobacierium wootlit in the presence of 5% CO.
[0013] Figure 4 describes. growth of Acetobacwrium woodii in the presence of 5% CO.
[0014] Figure 5 illustrates CO-, conversions, H2 conversions and cell density of Acelobacteriumwoodil at. pH 5.2 without a chelating agent (MTA) in the growth medium, [0015] Figure 6 describes growth ofAcetobacteriwn wood:// using ethylenediamine diacetic acid (EDDA) as a. chelating (complexing) agent in the growth medium.
[0016] Figure 7 illustrates the effect of molybdenum on acetic acid production by Acetobacterium [0017] Figure 8 illustrates the effect of molybdenum on gas flow rate requirement and cell density of Acelobacterium woodii.
DETAILED DESCRIPTION
[0018] The following description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles of exemplary embodiments.
The scope of the disclosure Should be determined with reference to the claims.
Definitions.
[000] Unless otherwise defined, the following terms as used. throughout this specification for the present disclosure are defined as follows and can include either the singular or plural forms of definitions below defined:
[0020] The term "about" modifying any amount refers to the variation in that amount encountered in real world conditions., e.g., in the lab, pilot plant, or production facility.
For example, an amount of an ingredient or measurement employed in a mixture or quantity when modified by "about" includes the variation and degree of care typically employed in measuring in an experimental condition in production plant or lab.
For example, the amount of a component of a product when modified by "about"
includes the variation between batches in multiple experiments in the plant or lab and the -variation 'inherent in the analytical method. Whether or not modified by "about;' the amounts include equivalents to those amounts_ Any quantity stated herein and modified by "about" can .also be employed in the present disclosure as the amount not modified by "about".
[0021] The term. "fermentor" includes a fermentation deyicelbioreactor consisting of one or more vessels and/or towers or piping arrangements, Which includes a batch reactor, semi-batch reactor, continuous .reactor, continuous stirred tank reactor (CSTR), bubble column reactor., external circulation loop reactor, internal circulation loop reactor, immobilized cell reactor (ICR) trickle bed reactor (TBR), moving bed biofilm reactor (MBBR)., gas lift reactor, membrane reactor such as hollow fibre membrane bioreactor (HFMBR), static mixer, gas lift fennentor, or other vessel or other device suitable for gas-liquid contact.
[0022] The terms "fermentation", fermentation process" or "fermentation reaction" and the like are intended to encompass both the growth phase and product biosynthesis phase of the process. In one aspect, fermentation refers to conversion of C07 to acetic acid.
[0023] The term "cell density" means mass of microorganism cells per 'unit volume of fermentation broth, for example,. grams/liter.
[0024] The term "specific CO2 uptake means an amount of C07 in mmoles consumed by unit mass of microorganism cells (g) per unit time in minutes, i.e.
mmoletgramlin [0025] As used herein, productivity is expressed as STY. In this aspect, alcohol productivity may be expressed as STY (space time yield expressed as g ethanoll(Liday) or (g acetic acid I(L-day).
CO2-Containing Gaseous Substrate [0026] In one aspect:, the process includes providing a CO2-containing gaseous substrate to .a bioreactor. A CO2-containing substrate may include any gas that includes CO2. In this aspect, a C07-containing gas may include industrial gases, fermentor gas streams including for example, fermentor off-gases and mixtures thereof. In a related aspect, the CO2-containing substrate may include hydrogen or it may be blended with a 'hydrogen source to provide desired levels and ratios of FI7 to C07.
[0027] Industrial gases: In one aspect, the process includes providing a CO2-containing gaseous substrate to a bioreactor where the C0.2-containing gaseous substrate is generated from industrial gases.. Some examples of industrial gases include steel mill gas, industrial flue gas and incinerator exhaust gas. Examples of industrial gases include gases produced during ferrous metal products manufacturing, non-ferrous products manufacturing; petroleum refining processes, gasification of coal, gasification of.
biomass, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. Sources of hydrogen may include fossil fuels, steam reforming, oxidation of methane, coal gasification, and water electrolysis..
[00.28] Depending on the composition of the gaseous CO2-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known method& Further, depending on the composition of the gaseous CO2-containing substrate, the process may include adjusting the CO2-containing substrate to increase or decrease concentrations of CO2 and/or H2 to fall within desired ranges.
[0029] Fermentor Gas Streams:. In one aspect, the process includes providing a containing substrate to a. bioreactor where the CO2-containing substrate is a fermentor gas stream. Some examples of fermentor gas streams include fermentor off-gas generated in the fermentation of syngas. Some examples of syngas fermentation are described in U.S.
Patent No. 7,285,402, filed July 23, 2001, which is incorporated -herein by reference.
[0030] In one aspect, the process has applicability to supporting the production of alcohol from gaseous substrates such as high volume. CO-containing industrial flue gases. In some aspects, a gas that includes CO is derived from carbon containing waste, for example, industrial waste gases or from the gasification of other wastes. The fermentation of CO-containing gas may result in CO2 in fermentor off-gas. As such, the processes represent effective processes for capturing carbon that would otherwise be exhausted into: the environment. In this aspect, the off-gas from the fermentation of CO-containing gas may include about 0.5 mole % to about 50 mole .% CO.
.[0031] Blending of gas streams: According to particular aspects, streams from two or more sources can be combined and/or blended to produce a desirable and/or optimized substrate. stream. For example, a stream comprising a. high concentration of .CO2, such as the exhaust from a steel mill, can be combined with a stream comprising high concentrations of H2, such as the off-gas from a steel -min coke oven.
[0032] Depending on the composition of the CO2-containing substrate, the CO2-containing substrate may be provided directly to a fermentation process or may be further modified to include an appropriate H2 to CO2 molar ratio. The CO2-containing substrate may include from about 5 to about 90 mole % CO2 and from. about 5 to about 90 mole %
H2. 14 one aspect, the CO?. containing gas stream includes about. 5 to about 66.6% CO?.
[0033] In another aspect, the CO2-containing substrate may include from about 0 mole %
to about 50. mole % CO., in another aspect, about 0.5 mole % CO to about 50 mole % CO, inanother aspect, .about 0.5 mole % CO to about 5 mole % CO, and in another aspect, about 2 mole .% CO to about .5 mole % CO.
100341 In one aspect, the acetogenic bacteria will have a molar ratio of consumption of H2 to CO2 at a. ratio of about 4:1 to about 1:2. Hence, any substrate gas provided to the.
bioreactor that includes H2 and CO2 can be utilized. However, optimal levels of substrate gas provided to the bioreactor will have a ratio of H? to CO2 of about 4:1 to about 1:1, in another aspect, about 2:1, and in another aspect, about 3,5:1 to about 1.5:1.
Bioreactor Design and Operation [0035] Descriptions of fermentor designs are described in U.S. Serial Nos.
13/471,827 and 13/471,858, both filed May 15, 2012, and U.S. Serial No. 13/473,167, filed May 16, 2012, all of which are incorporated herein by reference.
[0036] The fermentation should desirably be carried out under appropriate conditions for the desired fermentation to occur (e.g. C0?-to-acetic acid), Reaction conditions to considered include pressure, temperature, gas flow rate, liquid flow rate, medium pH, agitation rate (if 'using a stirred tank reactor), inoculum level, and maximum acetic acid concentration to avoid product inhibition. In this aspect, the process includes reaction conditions in the following ranges:
Pressure: about 0 to about 500 psi;
Temperature: about 30 C to about 42 'C.;.
Medium pH: about 4 to about 6.9;
Agitation rate: about 100 to about 2000 rpm;
Nutrient supply as described herein.
Acetogenic Bacteria [0037] In one aspect, the microorganisms utilized include acetogenic bacteria that 'include a sodium pump which may also be described as sodium-translocating ATPases (for membrane bioenergetics).. Sodium-translocating ATPase are described in Muller, "Energy Conservation in Acetogenic Bacteria", Appl. Environ. I'vlicrobiol.
November 2003, vol. 69, no. 1.1, pp. 6345-63.53, which is incorporated herein by reference. The term sodium translocating ATPase may be used interchangeably with sodium dependent ATPase. Acetogens that include a sodium-translocating ATPase require about 500 ppm NaCt in their growth medium for growth. To determine if an acetogen includes a sodium-translocating ATPase, the acetogen is inoculated into a serum bottles containing about 30 to about 50 ml of growth medium with about 0 to about 2000 ppm NaCl.
Growth at NaCl concentrations of about 500 ppm or more means that the acetogen includes a sodium-translocating ATPase.
[0038] In this aspect, suitable microorganisms include Ace 1 obacierium bacteria, Acetogenium kivul, Acewanaerobilim noterote. 4cetobacerium woodii, Alkalibaculum bacehi CP11 (ATCC BAA-1772), Moore/Ia thermoacetko.õ116orella thermoctutotropJzica,RomilmoccosptValtal,f$ and combinations thereof. In another aspect, the microorganism is Acetobacterium Medium Compositions and Control of Medium. Feed Rates [0039] In accordance with one aspect, the fermentation process is started by addition of a suitable medium. to the reactor vessel. The liquid contained in the reactor vessel may include any type of suitable nutrient medium or fermentation medium. The nutrient medium will Maude vitamins and minerals effective for permitting growth of the microorganism being. used. Sterilization may not always be required.
[0040] Concentrations of various medium components are as follows:.
Element Feed Rate Concentration rig/gram cells/min mg/L
NH4 + 82-32.80 20.5-820 Fe 0.85.-34 0.28-8..5 Ni 0.07.-2.81 0,023-0.702 Co 0.037-1.49 0.012-0.373 Se. 0:027-1.1 0.009-0.274 Zn. 0:59-23.8: 0.198-5.95 Mo 0.003-0.397 0.003-0.1 chelator 2.5-100 0,83-25 0.8732.1 0,26-8.03 98-3933 32.77-983.35 Mg 0.71-28.69 0.23-7.18 Na 875-35000 290-8750 15-625 2.08-62.5 20-805 6.7-201.3 c1-biotin 0,016-0.64 0.005-0.16 thiamine HCl 0.04-1:6 0.01-0.4 calcium-D-pantothenate 0.02-0,81 0.006-0_202 [0041] Vitamins solution contains d-biotin, thiamine HC1, and calcium-D-pantothenate.
[0042] 0.5 M NaOH was used to keep the pH around 5.55. The approximate usage of NaOH per gram of cells per hour was 0.1 to 0.4 ml/min per gram of cells.
[0043] Process operation maintains a pH in a range of about 4 to about 6:9, in another aspect, about 5 to about 6.5, in another aspect about 5.1 to about 6, and in another aspect, about 5.2 to about 6. The medium includes less than about 0.01 g/L yeast extract and less.
than about 0:01 g/L: carbohydrates.
[0044] The composition also includes a sodium ion concentration of about 40 to about 500 mmol per liter, in another aspect, about 40 to about 250 mmol per liter and in another aspect, a sodium ion concentration of about 50 to about 200 .mmol per liter.
In one aspect, the sodium ion concentration is. about 500 ppm to about 8000 ppm, in another.
aspect, about 1000 ppm to about 7000 ppm, in another aspect, about 3000 ppm.
to about 6000 ppm, in another aspect,. about 2000 to about 5000 ppm Na, and in another aspect, about 3000 to about 4000 ppm Na. The sodium ion source is provided by a compound selected from the group consisting of sodium chloride, sodium hydroxide, sodium phosphate, sodium sulfate, sodium nitrate, sodium bicarbonate, sodium bisulfate and mixtures thereof [0045] The composition includes a source of molybdenum. In this aspect the molybdenum concentration is about 3.97 1.tg/L to about 396.5 14g/L, and in another aspect, about 7.93 ug/L to about 198:25 tg/L. Sources of molybdenum include Na2Mo04, CaMo04, FeMoai and mixtures thereof [0046] The composition may also include a compiexing agent In this aspect, a complexity agent may be included in the composition when the composition has a pH of about 5.2 or greater. The complexing agent may include ethylenediaminetetraacetic acid (EDTA),..ethylenediamine diacetic acid (EDDA), ethylenediamine disuccinic acid (EDDS) and mixtures thereof.
[0047] The composition may include one or more of a source of NH4, P, K, Fe, Ni, Co, .Se, Zn., or Mg. Sources of each of these elements may be as follows.
[0048] NH4: The nitrogen may be provided from a nitrogen source selected from the group consisting of ammonium hydroxide, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, and mixtures thereof.
[0049] P: The phosphorous may be provided from a phosphorous source selected from the group -consisting of phosphoric acid, ammonium phosphate, potassium phosphate, and mixtures thereof [0050] K: The potassium may be provided from a potassium source selected from the group consisting of potassium- chloride, potassium phosphate, potassium nitrate,.
potassium sulfate, and mixtures. thereof:
[0051] Fe: The iron may be provided from an iron source selected from the group consisting of ferrous chloride, ferrous sulfate, and mixtures thereof [0052] Ni: The nickel may be provided from a nickel source selected from the group consisting of nickel chloride, nickel sulfate, nickel nitrate, and mixtures thereof [0053] Co: The cobalt may be provided from a cobalt source selected from the group consisting of cobalt chloride, cobalt -fluoride, cobalt bromide, cobalt iodide, and mixtures thereof [0054] Se: The selenium may be provided from Na2Se03. C3-16NG2Se, and mixtures thereof [0055] Zn: The zinc may be provided from ZnSO4.
[0056] W: The tungsten may be provided from a tungsten source selected from the group consisting of sodium tungstate, calcium tungstate, potassium tungstate, and mixtures thereof
100111 Figure 2 illustrates acetic acid production by Acetobacterium wood/i.
[0012] Figure 3 describes growth of Acetobacierium wootlit in the presence of 5% CO.
[0013] Figure 4 describes. growth of Acetobacwrium woodii in the presence of 5% CO.
[0014] Figure 5 illustrates CO-, conversions, H2 conversions and cell density of Acelobacteriumwoodil at. pH 5.2 without a chelating agent (MTA) in the growth medium, [0015] Figure 6 describes growth ofAcetobacteriwn wood:// using ethylenediamine diacetic acid (EDDA) as a. chelating (complexing) agent in the growth medium.
[0016] Figure 7 illustrates the effect of molybdenum on acetic acid production by Acetobacterium [0017] Figure 8 illustrates the effect of molybdenum on gas flow rate requirement and cell density of Acelobacterium woodii.
DETAILED DESCRIPTION
[0018] The following description is not to be taken in a limiting sense, but is made merely for the purpose of describing the general principles of exemplary embodiments.
The scope of the disclosure Should be determined with reference to the claims.
Definitions.
[000] Unless otherwise defined, the following terms as used. throughout this specification for the present disclosure are defined as follows and can include either the singular or plural forms of definitions below defined:
[0020] The term "about" modifying any amount refers to the variation in that amount encountered in real world conditions., e.g., in the lab, pilot plant, or production facility.
For example, an amount of an ingredient or measurement employed in a mixture or quantity when modified by "about" includes the variation and degree of care typically employed in measuring in an experimental condition in production plant or lab.
For example, the amount of a component of a product when modified by "about"
includes the variation between batches in multiple experiments in the plant or lab and the -variation 'inherent in the analytical method. Whether or not modified by "about;' the amounts include equivalents to those amounts_ Any quantity stated herein and modified by "about" can .also be employed in the present disclosure as the amount not modified by "about".
[0021] The term. "fermentor" includes a fermentation deyicelbioreactor consisting of one or more vessels and/or towers or piping arrangements, Which includes a batch reactor, semi-batch reactor, continuous .reactor, continuous stirred tank reactor (CSTR), bubble column reactor., external circulation loop reactor, internal circulation loop reactor, immobilized cell reactor (ICR) trickle bed reactor (TBR), moving bed biofilm reactor (MBBR)., gas lift reactor, membrane reactor such as hollow fibre membrane bioreactor (HFMBR), static mixer, gas lift fennentor, or other vessel or other device suitable for gas-liquid contact.
[0022] The terms "fermentation", fermentation process" or "fermentation reaction" and the like are intended to encompass both the growth phase and product biosynthesis phase of the process. In one aspect, fermentation refers to conversion of C07 to acetic acid.
[0023] The term "cell density" means mass of microorganism cells per 'unit volume of fermentation broth, for example,. grams/liter.
[0024] The term "specific CO2 uptake means an amount of C07 in mmoles consumed by unit mass of microorganism cells (g) per unit time in minutes, i.e.
mmoletgramlin [0025] As used herein, productivity is expressed as STY. In this aspect, alcohol productivity may be expressed as STY (space time yield expressed as g ethanoll(Liday) or (g acetic acid I(L-day).
CO2-Containing Gaseous Substrate [0026] In one aspect:, the process includes providing a CO2-containing gaseous substrate to .a bioreactor. A CO2-containing substrate may include any gas that includes CO2. In this aspect, a C07-containing gas may include industrial gases, fermentor gas streams including for example, fermentor off-gases and mixtures thereof. In a related aspect, the CO2-containing substrate may include hydrogen or it may be blended with a 'hydrogen source to provide desired levels and ratios of FI7 to C07.
[0027] Industrial gases: In one aspect, the process includes providing a CO2-containing gaseous substrate to a bioreactor where the C0.2-containing gaseous substrate is generated from industrial gases.. Some examples of industrial gases include steel mill gas, industrial flue gas and incinerator exhaust gas. Examples of industrial gases include gases produced during ferrous metal products manufacturing, non-ferrous products manufacturing; petroleum refining processes, gasification of coal, gasification of.
biomass, electric power production, carbon black production, ammonia production, methanol production and coke manufacturing. Sources of hydrogen may include fossil fuels, steam reforming, oxidation of methane, coal gasification, and water electrolysis..
[00.28] Depending on the composition of the gaseous CO2-containing substrate, it may also be desirable to treat it to remove any undesired impurities, such as dust particles before introducing it to the fermentation. For example, the gaseous substrate may be filtered or scrubbed using known method& Further, depending on the composition of the gaseous CO2-containing substrate, the process may include adjusting the CO2-containing substrate to increase or decrease concentrations of CO2 and/or H2 to fall within desired ranges.
[0029] Fermentor Gas Streams:. In one aspect, the process includes providing a containing substrate to a. bioreactor where the CO2-containing substrate is a fermentor gas stream. Some examples of fermentor gas streams include fermentor off-gas generated in the fermentation of syngas. Some examples of syngas fermentation are described in U.S.
Patent No. 7,285,402, filed July 23, 2001, which is incorporated -herein by reference.
[0030] In one aspect, the process has applicability to supporting the production of alcohol from gaseous substrates such as high volume. CO-containing industrial flue gases. In some aspects, a gas that includes CO is derived from carbon containing waste, for example, industrial waste gases or from the gasification of other wastes. The fermentation of CO-containing gas may result in CO2 in fermentor off-gas. As such, the processes represent effective processes for capturing carbon that would otherwise be exhausted into: the environment. In this aspect, the off-gas from the fermentation of CO-containing gas may include about 0.5 mole % to about 50 mole .% CO.
.[0031] Blending of gas streams: According to particular aspects, streams from two or more sources can be combined and/or blended to produce a desirable and/or optimized substrate. stream. For example, a stream comprising a. high concentration of .CO2, such as the exhaust from a steel mill, can be combined with a stream comprising high concentrations of H2, such as the off-gas from a steel -min coke oven.
[0032] Depending on the composition of the CO2-containing substrate, the CO2-containing substrate may be provided directly to a fermentation process or may be further modified to include an appropriate H2 to CO2 molar ratio. The CO2-containing substrate may include from about 5 to about 90 mole % CO2 and from. about 5 to about 90 mole %
H2. 14 one aspect, the CO?. containing gas stream includes about. 5 to about 66.6% CO?.
[0033] In another aspect, the CO2-containing substrate may include from about 0 mole %
to about 50. mole % CO., in another aspect, about 0.5 mole % CO to about 50 mole % CO, inanother aspect, .about 0.5 mole % CO to about 5 mole % CO, and in another aspect, about 2 mole .% CO to about .5 mole % CO.
100341 In one aspect, the acetogenic bacteria will have a molar ratio of consumption of H2 to CO2 at a. ratio of about 4:1 to about 1:2. Hence, any substrate gas provided to the.
bioreactor that includes H2 and CO2 can be utilized. However, optimal levels of substrate gas provided to the bioreactor will have a ratio of H? to CO2 of about 4:1 to about 1:1, in another aspect, about 2:1, and in another aspect, about 3,5:1 to about 1.5:1.
Bioreactor Design and Operation [0035] Descriptions of fermentor designs are described in U.S. Serial Nos.
13/471,827 and 13/471,858, both filed May 15, 2012, and U.S. Serial No. 13/473,167, filed May 16, 2012, all of which are incorporated herein by reference.
[0036] The fermentation should desirably be carried out under appropriate conditions for the desired fermentation to occur (e.g. C0?-to-acetic acid), Reaction conditions to considered include pressure, temperature, gas flow rate, liquid flow rate, medium pH, agitation rate (if 'using a stirred tank reactor), inoculum level, and maximum acetic acid concentration to avoid product inhibition. In this aspect, the process includes reaction conditions in the following ranges:
Pressure: about 0 to about 500 psi;
Temperature: about 30 C to about 42 'C.;.
Medium pH: about 4 to about 6.9;
Agitation rate: about 100 to about 2000 rpm;
Nutrient supply as described herein.
Acetogenic Bacteria [0037] In one aspect, the microorganisms utilized include acetogenic bacteria that 'include a sodium pump which may also be described as sodium-translocating ATPases (for membrane bioenergetics).. Sodium-translocating ATPase are described in Muller, "Energy Conservation in Acetogenic Bacteria", Appl. Environ. I'vlicrobiol.
November 2003, vol. 69, no. 1.1, pp. 6345-63.53, which is incorporated herein by reference. The term sodium translocating ATPase may be used interchangeably with sodium dependent ATPase. Acetogens that include a sodium-translocating ATPase require about 500 ppm NaCt in their growth medium for growth. To determine if an acetogen includes a sodium-translocating ATPase, the acetogen is inoculated into a serum bottles containing about 30 to about 50 ml of growth medium with about 0 to about 2000 ppm NaCl.
Growth at NaCl concentrations of about 500 ppm or more means that the acetogen includes a sodium-translocating ATPase.
[0038] In this aspect, suitable microorganisms include Ace 1 obacierium bacteria, Acetogenium kivul, Acewanaerobilim noterote. 4cetobacerium woodii, Alkalibaculum bacehi CP11 (ATCC BAA-1772), Moore/Ia thermoacetko.õ116orella thermoctutotropJzica,RomilmoccosptValtal,f$ and combinations thereof. In another aspect, the microorganism is Acetobacterium Medium Compositions and Control of Medium. Feed Rates [0039] In accordance with one aspect, the fermentation process is started by addition of a suitable medium. to the reactor vessel. The liquid contained in the reactor vessel may include any type of suitable nutrient medium or fermentation medium. The nutrient medium will Maude vitamins and minerals effective for permitting growth of the microorganism being. used. Sterilization may not always be required.
[0040] Concentrations of various medium components are as follows:.
Element Feed Rate Concentration rig/gram cells/min mg/L
NH4 + 82-32.80 20.5-820 Fe 0.85.-34 0.28-8..5 Ni 0.07.-2.81 0,023-0.702 Co 0.037-1.49 0.012-0.373 Se. 0:027-1.1 0.009-0.274 Zn. 0:59-23.8: 0.198-5.95 Mo 0.003-0.397 0.003-0.1 chelator 2.5-100 0,83-25 0.8732.1 0,26-8.03 98-3933 32.77-983.35 Mg 0.71-28.69 0.23-7.18 Na 875-35000 290-8750 15-625 2.08-62.5 20-805 6.7-201.3 c1-biotin 0,016-0.64 0.005-0.16 thiamine HCl 0.04-1:6 0.01-0.4 calcium-D-pantothenate 0.02-0,81 0.006-0_202 [0041] Vitamins solution contains d-biotin, thiamine HC1, and calcium-D-pantothenate.
[0042] 0.5 M NaOH was used to keep the pH around 5.55. The approximate usage of NaOH per gram of cells per hour was 0.1 to 0.4 ml/min per gram of cells.
[0043] Process operation maintains a pH in a range of about 4 to about 6:9, in another aspect, about 5 to about 6.5, in another aspect about 5.1 to about 6, and in another aspect, about 5.2 to about 6. The medium includes less than about 0.01 g/L yeast extract and less.
than about 0:01 g/L: carbohydrates.
[0044] The composition also includes a sodium ion concentration of about 40 to about 500 mmol per liter, in another aspect, about 40 to about 250 mmol per liter and in another aspect, a sodium ion concentration of about 50 to about 200 .mmol per liter.
In one aspect, the sodium ion concentration is. about 500 ppm to about 8000 ppm, in another.
aspect, about 1000 ppm to about 7000 ppm, in another aspect, about 3000 ppm.
to about 6000 ppm, in another aspect,. about 2000 to about 5000 ppm Na, and in another aspect, about 3000 to about 4000 ppm Na. The sodium ion source is provided by a compound selected from the group consisting of sodium chloride, sodium hydroxide, sodium phosphate, sodium sulfate, sodium nitrate, sodium bicarbonate, sodium bisulfate and mixtures thereof [0045] The composition includes a source of molybdenum. In this aspect the molybdenum concentration is about 3.97 1.tg/L to about 396.5 14g/L, and in another aspect, about 7.93 ug/L to about 198:25 tg/L. Sources of molybdenum include Na2Mo04, CaMo04, FeMoai and mixtures thereof [0046] The composition may also include a compiexing agent In this aspect, a complexity agent may be included in the composition when the composition has a pH of about 5.2 or greater. The complexing agent may include ethylenediaminetetraacetic acid (EDTA),..ethylenediamine diacetic acid (EDDA), ethylenediamine disuccinic acid (EDDS) and mixtures thereof.
[0047] The composition may include one or more of a source of NH4, P, K, Fe, Ni, Co, .Se, Zn., or Mg. Sources of each of these elements may be as follows.
[0048] NH4: The nitrogen may be provided from a nitrogen source selected from the group consisting of ammonium hydroxide, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, and mixtures thereof.
[0049] P: The phosphorous may be provided from a phosphorous source selected from the group -consisting of phosphoric acid, ammonium phosphate, potassium phosphate, and mixtures thereof [0050] K: The potassium may be provided from a potassium source selected from the group consisting of potassium- chloride, potassium phosphate, potassium nitrate,.
potassium sulfate, and mixtures. thereof:
[0051] Fe: The iron may be provided from an iron source selected from the group consisting of ferrous chloride, ferrous sulfate, and mixtures thereof [0052] Ni: The nickel may be provided from a nickel source selected from the group consisting of nickel chloride, nickel sulfate, nickel nitrate, and mixtures thereof [0053] Co: The cobalt may be provided from a cobalt source selected from the group consisting of cobalt chloride, cobalt -fluoride, cobalt bromide, cobalt iodide, and mixtures thereof [0054] Se: The selenium may be provided from Na2Se03. C3-16NG2Se, and mixtures thereof [0055] Zn: The zinc may be provided from ZnSO4.
[0056] W: The tungsten may be provided from a tungsten source selected from the group consisting of sodium tungstate, calcium tungstate, potassium tungstate, and mixtures thereof
11 [0057] Mg The magnesium may be provided from a magnesium source selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium phosphate, and mixtures thereof [0058] S: The composition may also, include sulfur_ The sulfur may be provided from a sulfur source selected from the group consisting of cysteine, sodium sulfide, NaHS,.
NaH7S and mixtures thereof.
Fermentation Startup and Post-Startup [0059] Startup: Upon inoculation, an initial feed gas supply rate is established effective for supplying the initial population of microorganisms. Effluent gas is analyzed to determine the content of the effluent. gas. Results of gas analysis are used to control feed gas rates. In this aspect, the process provides a minimal cell density of about 0.1 grams per liter. In another aspect, the process provides a calculated CO2 concentration (mmol/min) to initial .cell density ratio of about 0.05 to about 0.5, and in another aspect, about 0.01 to about I.
[0060] In one aspect, nutrients may be added to the culture to increase cell growth rates.
Suitable nutrients may include non-carbohydrate fractions of yeast extract.
[0061] Post-startup: Upon reaching desired levels, liquid phase and cellular material is withdrawn from the reactor and replenished with medium. The fermentation process is effective for increasing cell density as compared to a starting cell density_ In this aspect, the process provides an average cell density of about 2 to about 50 grams/liter, in another aspect, about 2 to about 30 grams/liter, in another aspect, about 2 to about 20 grams/liter, in another aspect, about 2 to about 10 grams/liter, and in another aspect, about 2 to about 6 grams/liter.
[0062] Production of Organic Acid: In another aspect, the process provides a source of Cl to C10 organic acids. In. this aspect, the process may include obtaining acid product:
or products from the fermentation liquid broth. In this aspect, provides a specific organic.
NaH7S and mixtures thereof.
Fermentation Startup and Post-Startup [0059] Startup: Upon inoculation, an initial feed gas supply rate is established effective for supplying the initial population of microorganisms. Effluent gas is analyzed to determine the content of the effluent. gas. Results of gas analysis are used to control feed gas rates. In this aspect, the process provides a minimal cell density of about 0.1 grams per liter. In another aspect, the process provides a calculated CO2 concentration (mmol/min) to initial .cell density ratio of about 0.05 to about 0.5, and in another aspect, about 0.01 to about I.
[0060] In one aspect, nutrients may be added to the culture to increase cell growth rates.
Suitable nutrients may include non-carbohydrate fractions of yeast extract.
[0061] Post-startup: Upon reaching desired levels, liquid phase and cellular material is withdrawn from the reactor and replenished with medium. The fermentation process is effective for increasing cell density as compared to a starting cell density_ In this aspect, the process provides an average cell density of about 2 to about 50 grams/liter, in another aspect, about 2 to about 30 grams/liter, in another aspect, about 2 to about 20 grams/liter, in another aspect, about 2 to about 10 grams/liter, and in another aspect, about 2 to about 6 grams/liter.
[0062] Production of Organic Acid: In another aspect, the process provides a source of Cl to C10 organic acids. In. this aspect, the process may include obtaining acid product:
or products from the fermentation liquid broth. In this aspect, provides a specific organic.
12 acid productivity of about 0_2 to about 50 grams organic acidlliter/dayig cells, in another aspect, about 0.2 to about 20 grams organic acid/literldaylg cells, in another aspect, about 10 to about 50 grams organic acid/liter/daylg cells, in another aspect, about 14 to about 30 grams organic acidlliter/daylg cells, in another aspect, about 2 to about 20 grams organic acidtliterlday/g cells and in another aspect, about 15 to about 25 grams organic acid/literidaylg .cells. In one aspect, the organic acid is acetic acid or butyric acid, or a mixture of both, [0063] Conversions of CO2 and H2: The process is effective for providing a CO2 uptake of about 0_05 to about 1.5 mmol CO2/minute/gram dry cells, an Hz uptake of about 0.08.
to about 1.5 mmol Hz/minute/gram dry cells,. The process is effective for providing about 25 to about 100% conversion of CO2, in another aspect, about. 50 to about 100%
conversion of CO2, and in another aspect, about 75 to about 100% conversion of CO2. In another aspect, the process is effective for providing about 25 to. about 100%
conversion of H2, in another aspect, about 50 to about 100% conversion of H2, and in another aspect, about 75 to about 100% conversion of I-I2.
[0064] Figure 1 shows a graph of CO2. conversion 104 and H2 conversion 102 by Acciobacteriumwoodil. A graphical illustration of acetic acid production 204 and its moving. average 202, and cell .density 206 versus time is shown in Figure 2, EXAMPLES
Example 1: Preparaiion of Acetobacierinin woodit [0065] An initial lyophilized pellet of Acelobacierium woodii was Obtained from German culture collection .DSIVIZ, strain .ID DSM-1030. Culture was initially revived from lyophilized pellet using rich medium (fructose and yeast extract). An adaptation method was used to remove fructose from serum bottle medium where concentration of fructose in growth medium was stepped down 75%, 50%, 10%, Growth rate and gas usage was used as an indicator of adaptation. (approximately 5 weeks). Preliminary pH.
adaptation 1.3 work in serum bottles reduced required pH from 7.4 to 6.0 (3 weeks). .At this point, culture was amplified and inoculated into a reactor. In a reactor culture was further adapted to grow in lower pH of 5.2 to 5.7.
Example 2: CSTR Reactor Startup Method for Acetobacterium woodil [0066] A synthesis gas containing CO, and 1-12 was continuously introduced into a stirred tank bioreactor containing Acetobacierium woodil, along with a liquid medium containing Vitamins, trace metals, cysteine (as sulfur .source), and .salts as described herein.
[0067] A New Brunswick Bioflow 310 reactor containing the fermentation medium was started with actively growing Acetobacterium wood/i. The rate of agitation of the reactor was set to 200 rpm. This agitation rate was increased throughout the experiment from 200 to 600 rpm. Feed gas flow to the reactor was increased from an initial at 49 mUmin to.
137 Temperature in the bioreactor was maintained at 33.5 C throughout the experiment. Samples of syngas feed into the bioreactor and off-gas from the bioreactor and fermentation broth in the bioreactor were taken at intervals, for example feed gas, off-gas and fermentation broth were sampled about daily, once two hours and once four hours respectively. Above samples were analyzed for consumption or production of various gas components, broth acetic acid concentration, and the optical density (cell density) of the culture. The unaroused volume of the reactor was maintained between 1600 to 1.750 ml throughout the experiment. Also the gas flow to the reactor was measured in real time by the mass flow controller regulating .syngas to the reactor. The feed .syngas composition was 70% H2, 25% CO2 and 5% N2. This experiment was concluded when stable operation was reached.
[0068] A cell recycle system (CRS) was attached to the reactor before the start of the experiment. During the experiment, the rate of flow of nutrients (growth medium) to the reactor was as indicated in the Table. Medium feed rate was maintained throughout the experiment. The base (NaOH) feed rate for pH control .was 0.14-0.44 nal/min, and through the CRS, 5.1. ¨ 5.4 ml/min permeate was drawn out from the reactor.
[0069] 112 and CO2 in the feed gas was fixed into cell material and acetic acid. The removal of H2 and CO2 was calculated by comparing inlet gas composition with the effluent gas composition. Component gas uptake is expressed in % of gas molecules converted by bacteria. In this experiment the following conversions were achieved;
40% - 54%, CO: 28% - 70%. In this experiment the rate of acetic acid production was 5-23 giliday.
[0070] Results can be summarized as follows:
Specific CO2 uptake (mmol C07/minlgram .:.Specific H2 uptake (nmol 1212/minigram dry cells) 0.20 047 ..
Acetic Acid productivity (g/t/day) Specific Acetic Acid productivity (g/Liday/gCellsr¨'' Average Cell Density (g/14 Example 3: Fermentation of COL, CO and 1-17 byAcetobacteriurn }moth/
[0071] A gas containing CO2 and 112 was continuously introduced into a stirred tank bioreactor containing Acetobacterium woodii, along with a conventional liquid medium containing vitamins, trace metals, and salts. Fermentations were started as described in Example 2 and then continued to stable operation. Mediums and process conditions are described in Example 2. In this Example, the feed gas included 5 mole % CO.
[0072] Figure 3 and Figure 4 describe growth of Acetobacterhmi woodii in the presence of 5% CO. Figure 3 illustrates cell density 302 and specific acetic acid productivity 304 versus time. Figure 4 illustrates H2: conversion 402, CO conversions 404, CO?
conversions 406, and cell density 408.
Example 4: Growth and Maintenance o TAcetobacterium woodil Culture at )11 5.2 without a Chelating Agent (EDTA) in the Growth Medium [0073] A gas stream containing CO2 and H, was continuously introduced into a stirred tank bioreactor containing Acetobacterium Plroodii, along with a growth medium as described herein.
[0074] A New. Brunswick Bioflow 11.5 reactor containing fermentation medium was started with actively growing Acetobacterium woodii (MV). The rate of agitation of the reactor was set to 6001pm. This agitation rate remained constant throughout the experiment. Feed gas flow to the reactor was maintained at 36.6mUmin to.
44.4mUmin.
Temperature in the bioreactor was maintained at 33 C throughout the experiment. Na+
levels were kept at 3500 to. 4000 ppm. Samples of gas feed into the -bioreactor and off-gas from the bioreactor and fermentation broth in the bioreactor were taken at intervals, for example feed gas, off-gas and fermentation broth was sampled about daily, once two.
hours and once four hours respectively. Above samples were analyzed for consumption or production of various gas components, broth acetic acid concentration, and the optical density (cell densitY). of the culture. The unaroused volume of the reactor was maintained between 1900 to 2275 ml throughout the experiment. Also the gas flow to the reactor was measured real time by the mass flow controller regulating syngas to the reactor_ The feed syngas composition of this experiment was 70% H,, 25% CO, and 5% N2.
[0075] A cell recycle system (CRS) was attached to the reactor before the start of the experiment. During the experiment, the rate of flow of nutrients (growth medium) to the reactor was maintained at 2.8 ml/min. Medium feed rate was maintained throughout the experiment. The average rate of base NaOH)( requirement to maintain pH at 5.2 was 0.075 milmin, and through the CRS, 2.9 mIlmin permeate was drawn out from the reactor.
[0076] H2 and CO, in the feed gas was fixed into cell material and acetic acid. The removal of H2 and CO2 was calculated by comparing inlet gas composition with the effluent gas composition. Component gas uptake can be expressed in of gas molecules converted by bacteria.
[0077] The following conversions were achieved:
H2: 28% to 54%
CO?: 40% to 59%
The rate of acetic acid production was 0.7949 (g/L/day) Average cell density of the culture was 1.9 g/1_, CO?. conversions 502, H2. conversions 504 and cell density 506 are shown in Figure 5..
Example 5: Use of EDDA in Growth Medium [0078] Fermentations were started as described in Example 2 and included the use of ethylenediamine diacetic acid (EDDA). as a chelating (complexing) agent, Chelating agents are employed to keep metals in solution as the solubility of some of the metals employed in AW medium decreases with the increasing pH. If the pH of the reactor broth is above pH .5_2, chelating agents are employed to provide sufficient amounts of nutrients to AW Figure 6 shows a representative 96 hr period of the experiment that.
'illustrates the ability to maintain cell density 602 while producing increasing concentrations of acetic acid 604.
Example 6: Effect of Molybdenum Removal. and Re-AM/ion on Cell Metabolism [0079] Fermentations were. started as. described. in Example 2 and then continued to stable operation. Molybdenum was removed from growth media and then re-added to the growth medium .after .acetic .acid productivity had dropped to75% of its starting concentration..
[0080] Figure 7 illustrates. acetic acid productivity 703 plotted against its media flow rate 705 with the vertical lines indicating the removal and re-addition of molybdenum to the growth medium.. Starting at about 810 cumulative hours, a downward trend of HAc was observed with the molybdenum removal occurring at about 795. cumulative hours.
This downward trend .decreased, plateaued and then was reversed into an upward trend in correspondence with the re-addition of molybdenum to the media at about 900 hours._ [0081] Figure 8 illustrates cell. density 801 and gas flow rate (GFR) 806 plotted against time with the vertical lines indicating the removal and re-addition of molybdenum to the growth medium.. Starting at about 840 cumulative hours, the required GFR was reduced with the molybdenum removal occurring at about 795 cumulative hours. This downward trend was reversed into an upward trend in correspondence with the return of molybdenum to the media at about 900 hours.
[0082] While the disclosure herein disclosed has been described by means of specific embodiments, examples and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from. the scope of the disclosure set forth in the claims.
to about 1.5 mmol Hz/minute/gram dry cells,. The process is effective for providing about 25 to about 100% conversion of CO2, in another aspect, about. 50 to about 100%
conversion of CO2, and in another aspect, about 75 to about 100% conversion of CO2. In another aspect, the process is effective for providing about 25 to. about 100%
conversion of H2, in another aspect, about 50 to about 100% conversion of H2, and in another aspect, about 75 to about 100% conversion of I-I2.
[0064] Figure 1 shows a graph of CO2. conversion 104 and H2 conversion 102 by Acciobacteriumwoodil. A graphical illustration of acetic acid production 204 and its moving. average 202, and cell .density 206 versus time is shown in Figure 2, EXAMPLES
Example 1: Preparaiion of Acetobacierinin woodit [0065] An initial lyophilized pellet of Acelobacierium woodii was Obtained from German culture collection .DSIVIZ, strain .ID DSM-1030. Culture was initially revived from lyophilized pellet using rich medium (fructose and yeast extract). An adaptation method was used to remove fructose from serum bottle medium where concentration of fructose in growth medium was stepped down 75%, 50%, 10%, Growth rate and gas usage was used as an indicator of adaptation. (approximately 5 weeks). Preliminary pH.
adaptation 1.3 work in serum bottles reduced required pH from 7.4 to 6.0 (3 weeks). .At this point, culture was amplified and inoculated into a reactor. In a reactor culture was further adapted to grow in lower pH of 5.2 to 5.7.
Example 2: CSTR Reactor Startup Method for Acetobacterium woodil [0066] A synthesis gas containing CO, and 1-12 was continuously introduced into a stirred tank bioreactor containing Acetobacierium woodil, along with a liquid medium containing Vitamins, trace metals, cysteine (as sulfur .source), and .salts as described herein.
[0067] A New Brunswick Bioflow 310 reactor containing the fermentation medium was started with actively growing Acetobacterium wood/i. The rate of agitation of the reactor was set to 200 rpm. This agitation rate was increased throughout the experiment from 200 to 600 rpm. Feed gas flow to the reactor was increased from an initial at 49 mUmin to.
137 Temperature in the bioreactor was maintained at 33.5 C throughout the experiment. Samples of syngas feed into the bioreactor and off-gas from the bioreactor and fermentation broth in the bioreactor were taken at intervals, for example feed gas, off-gas and fermentation broth were sampled about daily, once two hours and once four hours respectively. Above samples were analyzed for consumption or production of various gas components, broth acetic acid concentration, and the optical density (cell density) of the culture. The unaroused volume of the reactor was maintained between 1600 to 1.750 ml throughout the experiment. Also the gas flow to the reactor was measured in real time by the mass flow controller regulating .syngas to the reactor. The feed .syngas composition was 70% H2, 25% CO2 and 5% N2. This experiment was concluded when stable operation was reached.
[0068] A cell recycle system (CRS) was attached to the reactor before the start of the experiment. During the experiment, the rate of flow of nutrients (growth medium) to the reactor was as indicated in the Table. Medium feed rate was maintained throughout the experiment. The base (NaOH) feed rate for pH control .was 0.14-0.44 nal/min, and through the CRS, 5.1. ¨ 5.4 ml/min permeate was drawn out from the reactor.
[0069] 112 and CO2 in the feed gas was fixed into cell material and acetic acid. The removal of H2 and CO2 was calculated by comparing inlet gas composition with the effluent gas composition. Component gas uptake is expressed in % of gas molecules converted by bacteria. In this experiment the following conversions were achieved;
40% - 54%, CO: 28% - 70%. In this experiment the rate of acetic acid production was 5-23 giliday.
[0070] Results can be summarized as follows:
Specific CO2 uptake (mmol C07/minlgram .:.Specific H2 uptake (nmol 1212/minigram dry cells) 0.20 047 ..
Acetic Acid productivity (g/t/day) Specific Acetic Acid productivity (g/Liday/gCellsr¨'' Average Cell Density (g/14 Example 3: Fermentation of COL, CO and 1-17 byAcetobacteriurn }moth/
[0071] A gas containing CO2 and 112 was continuously introduced into a stirred tank bioreactor containing Acetobacterium woodii, along with a conventional liquid medium containing vitamins, trace metals, and salts. Fermentations were started as described in Example 2 and then continued to stable operation. Mediums and process conditions are described in Example 2. In this Example, the feed gas included 5 mole % CO.
[0072] Figure 3 and Figure 4 describe growth of Acetobacterhmi woodii in the presence of 5% CO. Figure 3 illustrates cell density 302 and specific acetic acid productivity 304 versus time. Figure 4 illustrates H2: conversion 402, CO conversions 404, CO?
conversions 406, and cell density 408.
Example 4: Growth and Maintenance o TAcetobacterium woodil Culture at )11 5.2 without a Chelating Agent (EDTA) in the Growth Medium [0073] A gas stream containing CO2 and H, was continuously introduced into a stirred tank bioreactor containing Acetobacterium Plroodii, along with a growth medium as described herein.
[0074] A New. Brunswick Bioflow 11.5 reactor containing fermentation medium was started with actively growing Acetobacterium woodii (MV). The rate of agitation of the reactor was set to 6001pm. This agitation rate remained constant throughout the experiment. Feed gas flow to the reactor was maintained at 36.6mUmin to.
44.4mUmin.
Temperature in the bioreactor was maintained at 33 C throughout the experiment. Na+
levels were kept at 3500 to. 4000 ppm. Samples of gas feed into the -bioreactor and off-gas from the bioreactor and fermentation broth in the bioreactor were taken at intervals, for example feed gas, off-gas and fermentation broth was sampled about daily, once two.
hours and once four hours respectively. Above samples were analyzed for consumption or production of various gas components, broth acetic acid concentration, and the optical density (cell densitY). of the culture. The unaroused volume of the reactor was maintained between 1900 to 2275 ml throughout the experiment. Also the gas flow to the reactor was measured real time by the mass flow controller regulating syngas to the reactor_ The feed syngas composition of this experiment was 70% H,, 25% CO, and 5% N2.
[0075] A cell recycle system (CRS) was attached to the reactor before the start of the experiment. During the experiment, the rate of flow of nutrients (growth medium) to the reactor was maintained at 2.8 ml/min. Medium feed rate was maintained throughout the experiment. The average rate of base NaOH)( requirement to maintain pH at 5.2 was 0.075 milmin, and through the CRS, 2.9 mIlmin permeate was drawn out from the reactor.
[0076] H2 and CO, in the feed gas was fixed into cell material and acetic acid. The removal of H2 and CO2 was calculated by comparing inlet gas composition with the effluent gas composition. Component gas uptake can be expressed in of gas molecules converted by bacteria.
[0077] The following conversions were achieved:
H2: 28% to 54%
CO?: 40% to 59%
The rate of acetic acid production was 0.7949 (g/L/day) Average cell density of the culture was 1.9 g/1_, CO?. conversions 502, H2. conversions 504 and cell density 506 are shown in Figure 5..
Example 5: Use of EDDA in Growth Medium [0078] Fermentations were started as described in Example 2 and included the use of ethylenediamine diacetic acid (EDDA). as a chelating (complexing) agent, Chelating agents are employed to keep metals in solution as the solubility of some of the metals employed in AW medium decreases with the increasing pH. If the pH of the reactor broth is above pH .5_2, chelating agents are employed to provide sufficient amounts of nutrients to AW Figure 6 shows a representative 96 hr period of the experiment that.
'illustrates the ability to maintain cell density 602 while producing increasing concentrations of acetic acid 604.
Example 6: Effect of Molybdenum Removal. and Re-AM/ion on Cell Metabolism [0079] Fermentations were. started as. described. in Example 2 and then continued to stable operation. Molybdenum was removed from growth media and then re-added to the growth medium .after .acetic .acid productivity had dropped to75% of its starting concentration..
[0080] Figure 7 illustrates. acetic acid productivity 703 plotted against its media flow rate 705 with the vertical lines indicating the removal and re-addition of molybdenum to the growth medium.. Starting at about 810 cumulative hours, a downward trend of HAc was observed with the molybdenum removal occurring at about 795. cumulative hours.
This downward trend .decreased, plateaued and then was reversed into an upward trend in correspondence with the re-addition of molybdenum to the media at about 900 hours._ [0081] Figure 8 illustrates cell. density 801 and gas flow rate (GFR) 806 plotted against time with the vertical lines indicating the removal and re-addition of molybdenum to the growth medium.. Starting at about 840 cumulative hours, the required GFR was reduced with the molybdenum removal occurring at about 795 cumulative hours. This downward trend was reversed into an upward trend in correspondence with the return of molybdenum to the media at about 900 hours.
[0082] While the disclosure herein disclosed has been described by means of specific embodiments, examples and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from. the scope of the disclosure set forth in the claims.
Claims (22)
1. A process comprising:
providing a gaseous substrate to a bioreactor, the gaseous substrate comprising CO2 and containing about.5 to about 90 mole % CO2;
providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources;
and fermenting the gaseous substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids;
wherein the acetogenic bacteria includes a sodium translocating ATPase that is active during fermentation in the bioreactor, wherein the fermentation broth includes less than about 0.01 grams per liter yeast extract, and less than about 0.01 grams per liter carbohydrate, wherein the sodium ions are provided with a sodium feed rate of about 290 to about 8750 µg/gram of cells/minute, and wherein the fermentation broth is maintained at a pH in a range of about 4 to about 6.9.
providing a gaseous substrate to a bioreactor, the gaseous substrate comprising CO2 and containing about.5 to about 90 mole % CO2;
providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources;
and fermenting the gaseous substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids;
wherein the acetogenic bacteria includes a sodium translocating ATPase that is active during fermentation in the bioreactor, wherein the fermentation broth includes less than about 0.01 grams per liter yeast extract, and less than about 0.01 grams per liter carbohydrate, wherein the sodium ions are provided with a sodium feed rate of about 290 to about 8750 µg/gram of cells/minute, and wherein the fermentation broth is maintained at a pH in a range of about 4 to about 6.9.
2. The process of claim 1 wherein the CO2 containing gaseous substrate is selected from.
the group consisting of industrial gases, fermentor gas streams and mixtures thereof,
the group consisting of industrial gases, fermentor gas streams and mixtures thereof,
3. The process of claim 1 wherein the acetogenic bacteria is selected from the group consisting of Acetobacterium bacteria, Acetogenium kivui, Acetoanaeroblum noterae Acetobacterium woodii, AIkalibaculum bacchi CP11 (ATCC BAA-1772), Moorella thermoacetica, Moorella thermoautotrophica, Ruminococcus products, and combinations thereof.
4. The process of claim 3 wherein the acetogenic bacteria is Acetobacterium woodii.
5. The process of claim 1 wherein the sodium ion source is provided by a compound selected from the group consisting of sodium chloride, sodium hydroxide, sodium phosphate, sodium sulfate, sodium nitrate, sodium bicarbonate, sodium bisulfate and mixtures thereof.
6. The process of claim 1 wherein the organic acid is one or more C1 to C10 organic acid.
7. The process of claim 6 wherein the organic acid is acetic acid, butyric acid, or mixtures thereof.
8. A process. comprising:.
providing a gaseous substrate to a bioreactor, the gaseous substrate comprising and CO2 and H2 and containing about 5 to about 90 mole % CO2;
providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources;
and fermenting the gaseous substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids;
wherein the acetogenic bacteria includes a sodium translocating ATPase that is active during fermentation in the bioreactor, wherein the fermentation broth includes less than about 0.01 grams per liter yeast extract, less than about 0.01 grams per liter carbohydrate, wherein the sodium ions are provided with a sodium feed rate of about 290 to about. 8750 gg/gram of cells/minute, and wherein the fermentation broth is maintained at a pH in a range of about 4 to about 6.9.
providing a gaseous substrate to a bioreactor, the gaseous substrate comprising and CO2 and H2 and containing about 5 to about 90 mole % CO2;
providing acetogenic bacteria to the bioreactor;
providing sodium ions to the bioreactor through one or more sodium ion sources;
and fermenting the gaseous substrate with the acetogenic bacteria in a fermentation broth comprising the acetogenic bacteria and the one or more sodium ion sources to produce one or more organic acids;
wherein the acetogenic bacteria includes a sodium translocating ATPase that is active during fermentation in the bioreactor, wherein the fermentation broth includes less than about 0.01 grams per liter yeast extract, less than about 0.01 grams per liter carbohydrate, wherein the sodium ions are provided with a sodium feed rate of about 290 to about. 8750 gg/gram of cells/minute, and wherein the fermentation broth is maintained at a pH in a range of about 4 to about 6.9.
9, The process of claim 8 wherein the gaseous substrate is selected from the group consisting of industrial gases, fermentor gas streams and mixtures thereof.
10. The process of claim 8 wherein the acetogenic bacteria is selected from the group consisting of Acetobacterium bacteria, Acetogenium kivui, Acetoanaerobium noterae, Acetobacterium woodii, Alkalibaculum bacchi CP11 (ATCC BAA-1772), Moorella thermoacetica, Moorella thermoautotrophica, Ruminococcus productus, and combinations.
thereof.
thereof.
11. The process of claim 10 wherein the acetogenic bacteria is Acetobocterium woodii.
12. The process of claim 8 wherein the sodium ion source is provided by a compound selected from the group consisting of sodium chloride, sodium hydroxide, sodium phosphate, sodium sulfate, sodium nitrate, sodium bicarbonate, sodium bisulfate and mixtures thereof.
13. The process of claim 8 wherein the organic acid is one or more C1 to C10 organic acid.
14. The process of claim 13 wherein the organic acid is acetic acid, butyric acid, or mixtures thereof
15. A composition comprising:
one or more of a source of NH4+, P, K, Fe, Ni, Co, Se, Zn, W, or Mg;
about 875 to about 3.5,000 mg/L of a sodium ion source; and about 0.009 to about 0.397 mg/L of a Mo source, and wherein the composition includes less than about 0.01 grams per liter yeast.
extract, and less than about 0.01 grams per liter carbohydrates, and wherein the composition has a pH of about 4 to about 6.9.
one or more of a source of NH4+, P, K, Fe, Ni, Co, Se, Zn, W, or Mg;
about 875 to about 3.5,000 mg/L of a sodium ion source; and about 0.009 to about 0.397 mg/L of a Mo source, and wherein the composition includes less than about 0.01 grams per liter yeast.
extract, and less than about 0.01 grams per liter carbohydrates, and wherein the composition has a pH of about 4 to about 6.9.
16. The composition of claim 15 wherein the sodium ion source is provided by a compound selected from the group consisting of sodium chloride, sodium hydroxide, sodium phosphate, sodium sulfate, sodium nitrate, sodium bicarbonate, sodium bisulfate and mixtures thereof.
17. The composition of claim 15 wherein the composition includes a complexing agent selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), ethylenediamine diacetic acid (EDDA), ethylenediamine disuccinic acid (EDDS) and mixtures thereof
18. The composition of claim 15 wherein the composition comprises:
about 82 to about 3280 mg/L of a NH4+ source;
about 20.12 to about 805 mg/L of a phosphorous source; or about 98.33 to about 3933 mg/L of a potassium source.
about 82 to about 3280 mg/L of a NH4+ source;
about 20.12 to about 805 mg/L of a phosphorous source; or about 98.33 to about 3933 mg/L of a potassium source.
19., The composition of claim 18 wherein the nitrogen is provided from a nitrogen source selected from the group consisting of ammonium hydroxide, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, and mixtures thereof;
the phosphorous is provided from a phosphorous source selected from the group consisting of phosphoric acid, ammonium phosphate, potassium phosphate, and mixtures thereof; and the potassium is provided from a potassium source selected from the group consisting of potassium chloride, potassium phosphate, potassium nitrate, potassium sulfate, and mixtures thereof.
the phosphorous is provided from a phosphorous source selected from the group consisting of phosphoric acid, ammonium phosphate, potassium phosphate, and mixtures thereof; and the potassium is provided from a potassium source selected from the group consisting of potassium chloride, potassium phosphate, potassium nitrate, potassium sulfate, and mixtures thereof.
20. The composition of claim 19 wherein the composition comprises:
about 0.85 to about 34 mg/L of an iron source;
about 0.07 to about 2.81 mg/L of a nickel source;
about 0.037 to about 1.49 mg/L of a cobalt source;
about 0.027 to about 1.1 mg/L of a selenium source;
about 0.59 to about 23.8 mg/L of a zinc source;
about 80.25 to about 3210 mg/L of a tungsten source; or about 0.71 to about 28.69 mg/L of a magnesium source.
about 0.85 to about 34 mg/L of an iron source;
about 0.07 to about 2.81 mg/L of a nickel source;
about 0.037 to about 1.49 mg/L of a cobalt source;
about 0.027 to about 1.1 mg/L of a selenium source;
about 0.59 to about 23.8 mg/L of a zinc source;
about 80.25 to about 3210 mg/L of a tungsten source; or about 0.71 to about 28.69 mg/L of a magnesium source.
21. The composition of claim 20 wherein the iron is provided from an iron source selected from the group consisting of ferrous chloride, ferrous sulfate, and miXtures thereof;
the nickel is provided from a nickel source selected from the group consisting of nickel chloride, nickel sulfate, nickel nitrate, and mixtures thereof;
the cobalt is provided from a cobalt source selected from the group consisting of cobalt chloride, cobalt fluoride, cobalt bromide, cobalt iodide, and mixtures thereof;
the selenium is provided. from a selenium source selected from the group consisting of Na2SeO3, C3H6NO2Se, and mixtures thereof;
the zinc is provided from ZnSO4;
the tungsten is provided from a tungsten source selected from the group consisting of sodium tungstate, calcium tungstate, potassium tungstate, and mixtures thereof; and the magnesium is provided from a magnesium source selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium phosphate, and the sulfur is provided from a sulfur source selected from the group consisting of cysteine, sodium sulfide, and mixtures thereof.
the nickel is provided from a nickel source selected from the group consisting of nickel chloride, nickel sulfate, nickel nitrate, and mixtures thereof;
the cobalt is provided from a cobalt source selected from the group consisting of cobalt chloride, cobalt fluoride, cobalt bromide, cobalt iodide, and mixtures thereof;
the selenium is provided. from a selenium source selected from the group consisting of Na2SeO3, C3H6NO2Se, and mixtures thereof;
the zinc is provided from ZnSO4;
the tungsten is provided from a tungsten source selected from the group consisting of sodium tungstate, calcium tungstate, potassium tungstate, and mixtures thereof; and the magnesium is provided from a magnesium source selected from the group consisting of magnesium chloride, magnesium sulfate, magnesium phosphate, and the sulfur is provided from a sulfur source selected from the group consisting of cysteine, sodium sulfide, and mixtures thereof.
22. The composition of claim 15 wherein the sodium ion concentration is about 500 ppm to about 8000 ppm.
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